The Human Peroxisomal Targeting Signal Receptor, Pex5p, Is Translocated into the Peroxisomal Matrix and Recycled to the Cytosol  Vincent Dammai, Suresh Subramani  Cell  Volume 105, Issue 2, Pages 187-196 (April 2001) DOI: 10.1016/S0092-8674(01)00310-5

Figure 1 Constructs and Strategy used to Demonstrate Pex5p Shuttling (A) Fusion constructs of EGFP and Pex5p stably integrated into HeLa, Ala-T, and Bro-T cells. Relevant components and epitopes, and their order within the fusion proteins are indicated. (B) Strategy used to detect peroxisomal entry and exit of the fusion proteins using subcellular fractionations and immunoblotting with M1 and M2 antibodies. The diagram on the left represents the import into, and export out of, peroxisomes for the fusion proteins, while panels a, b, and c (right) show schematic gels of the expected subcellular distributions of the proteins in immunoblotting with M1 and M2 antibodies. Details are in the Results section Cell 2001 105, 187-196DOI: (10.1016/S0092-8674(01)00310-5)

Figure 2 Processing and Subcellular Locations of PTS2-GFP Fusions Untransfected cells or (A) HeLa, (B) Ala-T, or (C) Bro-T cells, expressing either PTS2wt-GFP or PTS2mut-GFP fusions, were subjected to differential centrifugation, and immunoblotted with M2 and anti-catalase antibodies. Open and solid arrows denote the unprocessed and processed forms of the fusion protein, respectively. Hom, cell homogenate Cell 2001 105, 187-196DOI: (10.1016/S0092-8674(01)00310-5)

Figure 3 Peroxisomal Entry of PTS2wt-GFP and Specificity of the Prethiolase Protease (A) Complementation of the PTS1 import defect in Ala-T cells coexpressing the PTS2-GFP and PTS2mut-Pex5p fusions. Differential centrifugation fractions were immunoblotted with M2 or anti-catalase antibodies. (B) Peroxisome-specific cleavage of the PTS2wt-GFP fusion by the prethiolase protease. Cell lysates expressing the constructs were immunoblotted with either M1 or M2 antibodies, as shown. Symbols are as in Figure 2 Cell 2001 105, 187-196DOI: (10.1016/S0092-8674(01)00310-5)

Figure 4 Pex5p Enters and Exits the Peroxisomal Matrix Independent of a Functional PTS2 Import Pathway Arrow symbols are as in Figure 2. (A) Expression levels and processing at the PPS of PTS2-Pex5p fusions in HeLa cells. Cell lysates at the indicated time points were immunoprecipitated with anti-Pex5p antibody and blotted with M2 antibody. (B) Subcellular fractions of HeLa, Ala-T, or Bro-T cells expressing PTS2mut-Pex5p were immunoblotted with M1 and anti-catalase antibodies Cell 2001 105, 187-196DOI: (10.1016/S0092-8674(01)00310-5)

Figure 5 The PTS2mut-Pex5p and PTS2wt-GFP Fusions Enter the Peroxisome Matrix, but Only the PTS2mut-Pex5p Fusion Recycles back to the Cytosol The fusion proteins, (A) PTS2mut-Pex5p and (B) PTS2wt-GFP, were coexpressed in HeLa, Ala-T, or Bro-T cells and the subcellular distributions of the processed and unprocessed forms of these fusion proteins were analyzed by immunoblotting of the same samples with M1 and M2 antibodies Cell 2001 105, 187-196DOI: (10.1016/S0092-8674(01)00310-5)

Figure 6 Protease Protection Assay Showing That a Portion of Peroxisome-Associated PTS2mut-Pex5p Is inside Peroxisomes Lanes 1–4 show the distribution, in subcellular fractions of HeLa cells, of peroxisomal matrix (catalase) and membrane markers (PMP-70) and of the unprocessed and processed forms of PTS2mut-Pex5p. Proteins in the pellet fractions were treated with Proteinase-K, with or without sonication and detergent (Triton X-100). Samples were subjected to SDS-PAGE and immunoblotting with appropriate antibodies Cell 2001 105, 187-196DOI: (10.1016/S0092-8674(01)00310-5)

Figure 7 Colocalization of the Processed Form of PTS2mut-Pex5p with Peroxisomes in HeLa Cells (A) Cells expressing the PTS2mut-Pex5p fusion were treated either with digitonin (25 μg/ml) alone (Experimental Procedures), to permeabilize only the plasma membrane (left panels), or with Triton X-100 (1%) to permeabilize both plasma and peroxisomal membranes (right panels). Indirect immunofluorescence was performed with antibodies against the peroxisomal matrix and membrane markers, catalase (left panels) and PMP-70 (right panels), respectively. (B) Indirect immunofluorescence with M1 (top left and bottom right panel), M2 (top right panel), and anti-PMP70 antibodies (bottom left panel) and colocalization of the processed form of PTS2mut-Pex5p with PMP70 (lower left and right panels) in cells permeabilized with Triton X-100. (C) Detection of the intraperoxisomal processed form of PTS2mut-Pex5p employing sequential permeabilization of the plasma membrane with digitonin (25 μg/ml), followed by washes to remove the cytosol, and subsequent permeabilization of the peroxisomal membrane with Triton X-100 (1%). Indirect immunofluorescence was performed with M1, M2, and anti-PMP70 antibodies. The central and right panels show colocalization in the same cells Cell 2001 105, 187-196DOI: (10.1016/S0092-8674(01)00310-5)

Figure 8 Pulse-Chase Analysis of Peroxisomal Import and Export of the PTS2mut-Pex5p Fusion Protein in HeLa Cells Conditions for the pulse-chase are in Experimental procedures. Subcellular fractions obtained at each time point were immunoprecipitated using M2 antibodies, and the immunoprecipitates were subjected to SDS-PAGE and autoradiography. (A) Autoradiogram showing the amount of processed and unprocessed forms of PTS2mut-Pex5p associated with subcellular fractions over the chase period. (B) Densitometric analysis of the pulse-chase results Cell 2001 105, 187-196DOI: (10.1016/S0092-8674(01)00310-5)